Recombinant DNA technology encompasses many methods for digesting and religating DNA fragments. One of the most widely known uses for recombinant DNA technology is insertion of new genes into target DNA fragments. During this procedure, the target fragment is typically digested with a restriction enzyme, such as EcoRI. Similarly, the insert DNA, having the gene of interest, is digested with the same enzyme. In one type of restriction enzyme digestion, cleavage of both the target DNA and insert DNA leaves overlapping 3' or 5' nucleotide fragments on each end. These overlapping fragments or "sticky ends" are well known properties of some restriction enzymes.
Incubation of the target and insert DNA together at an appropriate temperature allows the insert DNA to noncovalently bind to the target DNA. The target DNA and insert DNA are held together by hydrogen bonding of the "sticky ends". Further incubation with an enzyme, such as DNA ligase, results in ligation of the insert DNA to the target nucleotide strand.
Additional methods of directly cloning DNA fragments into target DNA sequences are available. One such method is described by Mead et al. (Bio/Technology (1991) 9:657). This method relies on the ability of Taq polymerase to inherently add deoxyadenosine (dATP) to the 3' end of some newly synthesized duplex molecules described in Clark, J. M. (1988) Nucleic Acids Research 20:9677. These single adenosine overhangs base pair with 3' thymidine (dTTP) overhangs at the insertion site of a specially designed vector. It has been found that even single base pairs are sufficient for hydrogen bonding two nucleotide sequences together.
Another method of adding an insert nucleotide fragment into a target DNA is known as blunt-end ligation. Digestion with some restriction enzymes, such as SrfI (GCCC/GGGC), SmaI (CCC/GGG), or Eco RV (GAT/ATC) do not leave any 3' or 5' overhanging nucleotides at the enzyme splice site. These enzymes are known as "blunt-end" enzymes due to this feature of their enzymatic activity. After digestion, blunt-end restriction enzymes maintain single 5' "terminal" phosphates on both sides of the restriction site. These terminal 5' phosphates are required by DNA ligase for any subsequent religation of the digested DNA sequence.
During ligation, DNA ligase covalently links hydrogen bonded double stranded DNA molecules. This enzyme requires a 5' terminal phosphate to act as an electron acceptor. This mechanism is explained in more detail below.
Synthesis of the phosphodiester bond between the 3' hydroxyl group of one nucleotidyl residue and the 5' phosphate ester of the adjacent group occurs in three stages. First, an adenylyl-enzyme intermediate forms. Either ATP or Nicotinamide Adenine Dinucleotide (NAD) can be the source of the adenylyl group. A covalent bond forms between an epsilon-amino group of a ligaselysyl residue and the phosphoryl group of AMP. Second, the 5'-terminal phosphate group of the DNA displaces ligase, resulting in an ADP-DNA adduct. Third, nucleophilic displacement by the same strand 3' hydroxyl group yields the final phosphodiester bond.
Knowledge of this phosphodiester bond synthesis mechanism is used by those with skill in the art. For instance, well known recombinant DNA techniques include the step of dephosphorylating a plasmid following restriction enzyme digestion to prevent religation of the cleaved ends (Sambrook et al. (1989) Molecular Cloning, A laboratory Manual; 2nd Ed. pp. 572 Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y.). In this method, the insert DNA sequence is cleaved leaving 5'-terminal phosphates on both sides of the restriction site. The target DNA sequence is then dephosphorylated with alkaline phosphatase, thereby being unable to religate due to its lack of 5' phosphates. If an insert DNA is added to this plasmid sample, it can hydrogen bond and ligate to the target due to its 5'-terminal phosphates.
Unfortunately, none of the aforementioned methods permit a researcher to choose a specific orientation for the inserted gene. This presents a distinct disadvantage in that the insert DNA can position itself in either of two 5'-3' orientations with respect to the target nucleotide sequence. Whereas in some instances the insert DNA orientation is unimportant, it is essential in certain procedures, such as ligating a gene to a promoter sequence for obtaining subsequent gene expression.
Following ligation of promoter and gene sequences, genes that are positioned in the wrong 5'.fwdarw.3' orientation will not transcribe a proper protein. Without any directionality provided for the insert, up to 50% of the genes, on average, will insert and ligate in the wrong orientation. This leads to a dramatic reduction in overall experimental efficiency. For this reason, many methods have been devised for preferentially cloning insert DNA fragments into target sequences in one orientation. These methods are commonly known as directional cloning techniques.
Initially, directional cloning was performed by digesting the target nucleotide sequences with two different restriction enzymes. This method resulted in a molecule with dissimilar DNA ends at the target insertion site. The insert DNA would then also be digested with the same two restriction enzymes thereby having two dissimilar DNA ends that corresponded to a specific orientation in the target insertion site. By following this procedure, the insert DNA could only bind the target sequence in one orientation.
Although this method has been widely used in the art, it does present drawbacks. For instance, digesting both the target DNA and insert DNA with multiple restriction enzymes is very time consuming. In addition, multiple enzyme digestions increase the risk that either the target or insert DNA sequence will be cleaved at an internal restriction site. Also, there are problems associated with digesting the ends of DNA strands, such as that required in cutting the insert sequence. Other problems in digesting DNA with two restriction endonucleases include having to change buffers between each reaction. This decreases the probability of properly cutting the DNA.
As discussed above, many investigators have attempted to improve methods relating to directionally cloning of DNA fragments. One of the most widely used procedures involving directional ligation relates to subcloning DNA fragments that have been amplified by the polymerase chain reaction (PCR).
PCR is a technique for amplifying specific regions of DNA by repeated rounds of synthesis and denaturing. In the first step of the technique primers are designed which flank the region to be amplified. A sample of the DNA sequence, in the presence of a molar excess of primers, is repeatedly incubated with polymerase and then the strands denatured. Following denaturation, a primer anneals the newly synthesized strands and the polymerization is repeated. This method leads to an exponential growth in the number of gene sequences.
In the past, directional cloning of PCR amplified DNA involved designing primers with specific internal restriction sites. The PCR fragments generated by these reactions would have primers on either end. Each primer contained its own cleavage sites. For example, following PCR amplification on the DNA of interest, the fragment is digested with two different restriction enzymes. This method leaves a PCR fragment with different restriction sites on each end. The fragment can then be specifically oriented in the target DNA sequence. However, this protocol has the same drawbacks as the aforementioned double digestion method. In addition, the PCR primers have more bases to accommodate the restriction site. This results in added expense for PCR primers.
Another method of directionally cloning an insert into a target sequence uses Exonuclease III (Kaluz, et al., Nucleic Acids Research, 20:16, pp. 4369-4370) to create the "sticky ends". In the method described by Kaluz et al., insert DNA fragments were digested with Exonuclease III. The number of nucleotides that Exonuclease III digests from the 3' end of DNA in a minute is well known. After a timed digestion, the insert fragments were left with 5' overlapping nucleotide tails. These tails were engineered so that the 5' ends would only hybridize in one orientation upon base pairing to the target plasmid DNA molecule.
However, the Exonuclease III method is very time dependent and enzyme continues to digest DNA as long as the reaction is incubated. For this reason, Exonuclease III might potentially digest through the end nucleotides and into the coding region of the insert DNA sequence, prompting an unwanted experimental result.
Another method uses the 3' exonuclease activity of T4 DNA polymerase to produce the sticky ends (Kuijper, J. L., et al. (1992) Gene 112:147-155.) One further method of directionally cloning PCR generated fragments into a target DNA sequence relies on incorporation of uracil into the PCR primers, followed by treatment with uracil-N-glycosylase (Nisson, P. C., et al. (1991) PCR Methods and Applications 1:120-123). The CLONEAMP.RTM. SYSTEM (Life Technologies Research Catalog (1992), Gaithersburg, Md.) utilizes uracil DNA glycosylase (UDG) to provide a method of directly cloning PCR fragments into a specially designed plasmid. PCR primers having internal deoxyuracil monophosphate (dUMP) nucleotides are used as primers to amplify the desired gene sequence. After PCR amplification, the sample is treated with UDG to remove the internal dUMP nucleotides. This enzymatic reaction leaves in 3'-overhangs on both ends of the PCR fragment. The fragment is then mixed with a vector having complementary "sticky ends" and ligated. Unfortunately, this method can be difficult to perform since it relies on specially designed dUMP primers for every reaction.
All of the above directional cloning methods require multiple restriction enzyme digestion, addition of extra nucleotides to the insert, or are expensive. For these reasons, there continues to exist a need for a simple, efficient, inexpensive method of directionally cloning a DNA fragment into its target sequence.